As is known, the identification of specific biological materials sequences is of significance in many areas including clinical, environmental and food microbiology diagnosis. In particular, the analysis of gene sequences plays a fundamental role in rapid detection of genetic mutations and infectious organisms. This means it is possible to make reliable diagnosis of diseases even before any symptoms appear.
Typical procedures for analyzing biological materials, such as nucleic acid, protein, lipid, carbohydrate, and other biological molecules, involve a variety of operations starting from raw material. These operations may include various degrees of cell separation or purification, lysis, amplification or purification, and analysis of the resulting amplification or purification product.
As an example, in DNA-based blood analyses samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells, which are generally not useful for DNA analysis. Then, the remaining white blood cells are broken up or lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed. Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
If RNA is to be analyzed, the procedures are similar, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof. A common analysis technique is an analysis by hybridization, wherein the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide detector fragments that are anchored on suitable substrates. The individual oligonucleotide detector fragments or “probes” may be complementary to the target amplified DNA strands. If the amplified DNA strands are complementary to the probes, stable bonds are formed between them (hybridization). The presence of a double stranded DNA in the mixture is thus indicative of a match and hybridization serves as a sequence detection mechanism.
In standard microarrays, the probes are attached to a solid surface (glass or silicon) using a linker molecule. Probe-target hybridization is usually detected and quantified by fluorescence-based detection. The hybridized detectors can be read using a wide variety of means, including optical, electromagnetic, electromechanical or thermal means.
Fluorescence-based detection, however, has several significant drawbacks, since: 1) it requires previous manipulation of the analyte to introduce the optical markers; 2) it requires expensive instrumentation for optical reading. Typical commercial equipments are systems with photomultiplier tubes (PMTs) or charge-coupled devices (CCDs), which consume high power and cannot be easily integrated with electronic circuits in an inexpensive way; and 3) its sensitivity may be limited by any lack of homogeneity in the marker distribution.
Recently, the use of quartz crystal microbalance (QCM) for hybridization detection has been proposed, which avoids the need for radioisotopes or fluorophores. Quartz is one member of a family of crystals that experience the piezoelectric effect (to generate an electric potential in response to applied mechanical stress), and the relationship between applied voltage and mechanical deformation is well known. A QCM measures a mass per unit area by measuring the change in frequency of a quartz crystal resonator, wherein the resonance is disturbed by the addition or removal of a small mass. The QCM can be used under vacuum, in gas phase, and more recently in liquid environments. In liquid, it is highly effective at determining the affinity of molecules to surfaces functionalized with recognition sites, and can be used to detect hybridization of nucleic acids, binding of peptides, and the like. Frequency measurements are easily made to high precision, thus it is easy to measure mass densities down to a level of below 1 μg/cm2.
FIGS. 1a and 1b show a quartz crystal microbalance 1 comprising a quartz disc 2 having gold electrodes 3a, 3b patterned on opposite sides of the quartz disc 2. One electrode 3a is covered by a sensing layer 4 capable of bonding with an analyte of interest (shown as dots 5 in FIG. 1b). The microbalance 1, connected in an oscillating circuit, fixes the oscillation frequency of the system at the natural frequency of the quartz disc 2.
By virtue of the hybridization, the mass of the quartz disc 2 increases and causes a variation in the oscillation frequency of the oscillating circuit, which can be easily measured.
FIG. 2 shows a quartz disc 2, functionalized by a biotinylated DNA probe layer having a high affinity with a previously deposited streptavidin layer. In particular, FIG. 2 shows a microbalance 1 wherein the sensing layer 4 comprises a plurality of single-strand DNA segments 7 functionalized by biotin 8; biotin 8 is bound to a streptavidin layer 9 deposited on the quartz disc 2. In the drawing, the single-strand DNA segment 7 is bound to a target strand 13, forming a double-strand 14.
The basic equation describing the relationship between the change in resonant frequency of an oscillating piezoelectric crystal and the mass deposited on the crystal surface was derived by Sauerbrey in 1959. Let A be the area of the quartz crystal in cm2, ΔM the mass difference due to the hybridization in g, f0 the rest resonance frequency of piezoelectric quartz crystal in MHz before hybridization, it can be obtained:
                              Δ          ⁢                                          ⁢          f                =                              -            2.26                    ×                      10                          -              6                                ⁢                                    f              o              2                        A                    ⁢          Δ          ⁢                                          ⁢          m                                    (        1        )            
The sensitivity S of the crystal sensor is given by:
                    S        =                                            Δ              ⁢                                                          ⁢              f                                      Δ              ⁢                                                          ⁢              m                                =                                    -              2.26                        ×                          10                              -                6                                      ⁢                                          f                o                2                            A                                                          (        2        )            
Thus, for a given piezoelectric crystal, the sensitivity of a microbalance can be increased by reducing the dimensions of the electrode surface. Therefore, miniaturization (e.g., silicon integration) will allow the QCM to reach very high sensitivity, so as to be able to detect even very small mass variations.
Semiconductor piezoelectric sensors have been disclosed for a plurality of applications. In particular, bulk-integrated, acoustic wave sensors using piezoelectric layers are known, wherein a piezoelectric layer, sandwiched between two electrode layers, overlies a cavity and forms an acoustic resonator (see, e.g., “Bulk Acoustic Wave Theory and Devices” Joel F. Rosenbaum Artech House Inc, 1988).
These electro-acoustic resonators have been proposed for forming sensors of several types, such as force, pressure, acceleration, weight and chemicals sensors, all of which exploit the variation in the oscillation frequency of the acoustic resonator following a mass variation and/or its geometrical configuration.
Known sensors have cavities formed by bulk micromachining by etching silicon substrate from the back using tetramethylammoniumhydroxide (TMAH, see, e.g. “Sensors and Microsystems: Proceedings of the 10th Italian Conference” A. G. Mignani, R. Falciai, C. Di Natale, A. D′Amico, World Scientific Publishing Company, July 2008—pages 326-331). In particular, according to this known technique, a silicon nitride layer, acting as an etch stop, is deposited on a surface of a silicon substrate. Then a stack of a first aluminum layer (lower electrode), an aluminum nitride layer and a second aluminum layer (upper electrode) is deposited. The substrate is anisotropically etched from the back and the etching stops at the silicon nitride layer. The wafer is then diced. In each die so obtained, the stack forms a diaphragm, whereon a thin layer of a sensitive probe material, such as porphyrin, may be deposited.
This process is not usual in current production lines for integrated circuits.
In addition, this etching technique causes the formation of a cavity having a trapezoidal section, with a shorter base formed by the diaphragm and sloping sides at 40°-60°. Since the thickness of the substrate is generally about 675-700 μm, the longer base of the cavity is longer about 1.2-1.4 mm than the diaphragm. The total area needed for each microbalance is thus much higher than the area of the oscillating region alone. Therefore, the microbalance is, as a whole, cumbersome.
As a consequence, in general, known piezoelectric sensors do not have wide application.
Thus, an aim of the invention is to devise a detector of biological materials that can be easily integrated, has high sensitivity, low manufacturing costs and high reliability.